Amino acid derivatives of diaryl 1,3,4-oxadiazolone

ABSTRACT

The present invention provides novel amino acid derivatives having the general formula ##STR1## wherein R 1 , R 2  and R 3  are as defined herein, or a nontoxic pharmaceutically acceptable salt or solvate thereof and are useful in the treatment of disorders which are responsive to the opening of potassium channels.

CROSS-REFERENCE TO RELATED APPLICATION

This is a nonprovisional application which claims the benefit ofprovisional application U.S. Ser. No. 60/072,925, filed Jan. 29, 1998.

FIELD OF THE INVENTION

The present invention is directed to novel amino acid derivatives of a1,3,4-oxadiazol-2(3H)-one compound which is a modulator of thelarge-conductance calcium-activated potassium (BK) channels and,therefore, useful in the protection of neuronal cells and diseasesarising from dysfunction of cellular membrane polarization andconductance. The present invention also provides a method of treatmentwith the novel substituted oxadiazolone derivatives and topharmaceutical compositions thereof.

BACKGROUND OF THE INVENTION

Stroke is presently recognized as the third leading cause of adultdisability and death in the United States and Europe. In the pastdecade, several therapeutic approaches for the minimization ofstroke-related brain damage have been pursued including inhibitors ofAMPA/kainate, N-methyl-D-aspartate (NMDA) and adenosine reuptakeinhibitors. It is the object of the present invention to provide novelcompounds that will modulate potassium channels, in particular,large-conductance calcium-activated potassium (BK) channels which willbe useful in reducing neuronal damage during ischemic conditions of astroke episode.

Potassium channels play a key role in regulation of cell membranepotential and modulation of cell excitability. Potassium channels arethemselves regulated by voltage, cell metabolism, calcium ion andreceptor mediated processes. [Cook, N. S., Trends in Pharmacol.Sciences, 9, pp.21-28 (1988); and Quast, U. and Cook, N. S., Trends inPharmacol. Sciences, 10, pp. 431-435 (1989)]. Calcium-activatedpotassium (K_(Ca)) channels are a diverse group of ion channels thatshare a dependence on intracellular calcium ions for activity. Theactivity of K_(Ca) channels is regulated by intracellular [Ca²⁺ ],membrane potential and phosphorylation. On the basis of theirsingle-channel conductances in symmetrical K⁺ solutions, K_(Ca) channelsare divided into three subclasses: large conductance (BK)>150 pS;intermediate conductance 50-150 pS; small conductance <50 pS. ("pS"stands for picosiemen, a unit of electrical conductance.)Large-conductance calcium-activated potassium (BK) channels are presentin many excitable cells including neurons, cardiac cells and varioustypes of smooth muscle cells. [Singer, J. J. and Walsh, J. V., PflugersArchiv., 408, pp. 98-111 (1987); Baro, I., and Escande, D., PflugersArchiv., 414 (Suppl. 1), pp. S168-S170 (1989); and Ahmed, F. et al., Br.J. Pharmacol., 83, pp. 227-233 (1984)].

Potassium ions play a dominant role in controlling the resting membranepotential in most excitable cells and in maintaining the transmembranevoltage near the K⁺ equilibrium potential (E_(k)) of about -90 mV. Ithas been shown that opening of potassium channels shifts the cellmembrane potential towards the equilibrium potassium membrane potential(E_(k)), resulting in hyperpolarization of the cell. [Cook, N. S.,Trends in Pharmacol. Sciences, 9, pp. 21-28 (1988]. Hyperpolarized cellsshow a reduced response to potentially damaging depolarizing stimuli. BKchannels which are regulated by both voltage and intracellular Ca²⁺ actto limit depolarization and calcium entry and may be particularlyeffective in blocking damaging stimuli. Therefore cell hyperpolarizationvia opening of BK channels may result in protection of neuronal cellsunder ischemic conditions.

The role of potassium channels in the operation of the smooth muscle ofthe human urinary bladder is discussed by S. Trivedi, et al. inBiochemical and Biophysical Research Communications, (1995), 213, No.2,pp. 404-409.

A range of synthetic and naturally occurring compounds with BK openingactivity have been reported. The avena pyrone extracted from avenasativa-common oats has been identified as a BK channel opener using alipid bi-layer technique [International Patent application WO 93/08800,published May 13,1993]. The flavanoid, Phloretin has been found toaffect the opening of Ca²⁺ -activated potassium channels in myelinatednerve fibers of Xenopus laevis using outside-out patches [Koh, D-S., etal., Neuroscience Lett., 165, pp.167-170 (1994)].

U.S. Pat. No. 3,971,803 issued to S. Rosenberger and K. Schwarzenbach onJul. 27, 1976, relates to compounds of Formula (i): ##STR2## wherein R₁is alkyl, cycloalkyl or aralkyl;

R₂ is hydrogen or R₁ ;

R₃ is hydrogen or C₁₋₄ alkyl;

Y and Z are independently O or S;

R₄ is either (1), if m=1, C₁₋₈ alkylene, --C_(x) H_(2x) --Q--C_(y)H_(2y) --(Q is O or S, x and y are integers whose sum is 2 to 4),phenylene, diphenylene or ##STR3## group; or (2) if m=2, alkylene,alkylene ether, alkylene thioether, diphenylene, or napthalene. Thecompounds are antioxidants for organic polymers.

EPO 0-533276-A1 published on Mar. 24, 1993, shows compounds of Formula(ii): ##STR4## wherein one of P or Q is an ortho-substituted phenylgroup and the other a substituted benzyl. The Formula (ii) compounds aremiticides and insecticides.

A. E. Wilder Smith disclosed in Arzneim. Forsch. (1967) 67, No.17, pp.768-772, the preparation and study of compounds of Formula (iii):##STR5## wherein X is H or Cl and n is 1 or 2. The compounds havetuberculostatic properties. Formula (iii) compounds do not encompasssubstitution para to the hydroxyl group.

J. L. Romine, et al. in International Patent Application WO 98/04135,published Feb. 5, 1998, describe a series of diphenyl heterocycles ofthe Formula (iv): ##STR6## wherein Het is a heterocyclic moiety selectedfrom inter alia, oxadiazolone. The compounds are useful as modulators ofthe large conductance calcium-activated potassium channels and thestarting material for the preparation of the compounds of the presentinvention is described therein wherein Het is 1,3,4-oxadiazol-2(3H)-one,m=1 and n=0, R^(c) is chloro, R^(d) is trifluoromethyl and R^(a) =R^(b)=R^(e) is hydrogen.

None of these references teach or suggest the novel compounds of thepresent invention.

SUMMARY OF THE INVENTION

The present invention provides novel amino acid derivatives of1,3,4-oxadiazolone having the general formula ##STR7## wherein R¹, R²and R³ are as defined below, or a nontoxic pharmaceutically acceptablesalt or solvate thereof. The present invention also providespharmaceutical compositions comprising said amino acid derivatives andto the method of treatment of disorders sensitive to potassium channelopening activity such as ischemia, stroke, convulsions, epilepsy,asthma, irritable bowel syndrome, migraine, traumatic brain injury,spinal cord injury, sexual dysfunction, and urinary incontinence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel amino acid derivatives of3-[(5-chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-onewhich is a potent opener of the large conductance, calcium-activated K⁺-channels (Bk channel) and the novel derivatives have the generalFormula I. ##STR8## wherein R¹ is hydrogen or methyl;

R² is hydrogen; or substituted or unsubstituted C₁₋₄ alkyl in which thesubstituent is selected from the group consisting of hydroxy, amino,methylthio, carboxyl, carboxamide, guanidino, phenyl or hydroxyphenyl;and

R³ is hydrogen or methyl; or R³ and R² are joined together to form apyrrolidinyl ring;

or a nontoxic pharmaceutically acceptable salt or solvate thereof.

The present invention also provides a method for the treatment of orprotection from disorders which are mediated by opening of the largeconductance calcium-activated K⁺ channels (BK channels) in a mammal inneed thereof, which comprises administering to said mammal atherapeutically effective amount of a compound of Formula I or anontoxic pharmaceutically acceptable salt thereof. Preferably, thecompounds of Formula I are useful in the treatment of ischemia, stroke,epilepsy, convulsions, asthma, irritable bowel syndrome, migraine,traumatic brain injury, spinal cord injury, sexual dysfunction, andurinary incontinence and other disorders sensitive to BK channelactivating activity.

The term "C₁₋₄ alkyl" as used herein and in the claims (unless thecontext indicates otherwise) means straight or branched chain alkylgroups such as methyl, ethyl, propyl, isopropyl, butyl and isobutyl.

The term "a nontoxic pharmaceutically acceptable salt" as used hereinand in the claims is intended to include nontoxic acid and base salts,and salts of zwitterionic species. Salts with a base is intended toinclude inorganic metallic salts such as sodium, potassium, calcium andmagnesium, the ammonium salt, and salts with nontoxic amines such astrialkylamines, pyridine, picoline, dibenzylamine, ethanolamine,N-methylmorpholine and other amines which have been used to form saltsof carboxylic acids. Suitable salts with an acid is intended to includeinorganic acid salts such as hydrochloride, hydrobromide, hydroiodide,sulfate, phosphate, and the like, and organic acid salts such asformate, acetate, maleate, citrate, succinate, ascorbate, lactate,fumarate and tartrate which have been used to form salts of basicamines.

Unless otherwise specified, the term "amino acid" derivative as usedherein means a naturally occurring amino acid. Suitable amino acids arethose described herein and other known amino acids such as alanine,glycine, arginine, cysteine, isoleucine, leucine, lysine, valine,methionine, phenylalanine, threonine and the like. It should beappreciated by those skilled in the art that the compounds of Formula Iwherein R¹ and R² contain an acyl residue of a naturally occurring aminoacid may be active in vivo as a prodrug, i.e., the amino acid residuemay be hydrolyzed by peptidase enzymes in the host to produce a moreactive form of the desired 1,3,4-oxadiazoline. Also, it should beappreciated by those skilled in the art that unnatural amino acids suchas those in the D-configuration may be substituted for the naturaloccurring amino acids in the L-configuration.

Generally, pharmaceutically acceptable salts of the invention are thosein which the counter anion does not contribute significantly to thetoxicity or pharmacological activity of the salt. In some instances,they have physical properties which make them more desirable forpharmaceutical formulations, such as solubility, lack of hygroscopicity,compressibility with respect to tablet formation and compatibility withother ingredients with which the substance may be used forpharmaceutical purposes. The salts are routinely made by admixture of aFormula I compound with the selected acid, preferably by contact insolution employing an excess of commonly used inert solvents such aswater, ether, dioxane, methylene chloride, isopropanol, methanol,ethanol, ethyl acetate and acetonitrile. They may also be made bymetathesis or treatment with an ion exchange resin under conditions inwhich the appropriate ion of a salt of the substance of the Formula I isreplaced by another ion under conditions which allow for separation ofthe desired species such as by precipitation from solution or extractioninto a solvent, or elution from or retention on an ion exchange resin.

Certain compounds of the present invention including thepharmaceutically acceptable salts thereof can exist as solvated formsincluding hydrated forms such as monohydrate, dihydrate, hemihydrate,trihydrate, tetrahydrate and the like. The products may be truesolvates, while in other cases, the products may merely retainadventitious solvent or be a mixture of solvate plus some adventitioussolvent. It should be appreciated by those skilled in the art thatsolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention.

In the method of the present invention, the term "therapeuticallyeffective amount" means the total amount of each active component of thecomposition that is sufficient to show a meaningful patient benefit,i.e., healing of acute conditions characterized by openers of largeconductance calcium-activated K⁺ channels or increase in the rate ofhealing of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously. Theterms "treat, treating, treatment" as used herein and in the claimsmeans preventing or ameliorating diseases, tissue damage and/or symptomsassociated with dysfunction of cellular membrane polarization andconductance.

In another aspect, this invention provides water-soluble prodrugs of3-[(5-chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-onewhich is described in WO 98/04135. As used herein the term prodrugdenotes a derivative of an active drug which is converted afteradministration back to the active drug. More particularly, it refers toamino acid derivatives of 1,3,4-oxadiazol-2(3H)-one compounds which maybe active drugs and/or which are capable of undergoing hydrolysis of theester or cleavage of the ester so as to release active free drug. Thephysiologically hydrolyzable groups serve as prodrugs by beinghydrolyzed in the body to yield the parent drug per se, and thus, thewater-soluble prodrugs of the present invention are preferred foradministration of the parent drug.

In still another aspect, this invention provides a method for thetreatment of or protection from disorders which are mediated by openingof the large conductance calcium-activated K⁺ channels (BK channels) ina mammal in need thereof, which comprises administering to said mammal atherapeutically effective amount of a compound of Formula I or anontoxic pharmaceutically acceptable salt, solvate or hydrate thereof.Preferably, the compounds of Formula I are useful in the treatment ofischemia, stroke, convulsions, epilepsy, asthma, irritable bowelsyndrome, migraine, traumatic brain injury, spinal cord injury, urinaryincontinence and sexual dysfunction in both men (erectile dysfunction,for example, due to diabetes mellitus, spinal cord injury, radicalprostatectomy, psychogenic etiology or any other cause) and women byimproving blood flow to the genitalia, especially the corpus cavernosum,and other disorders sensitive to BK channel activating activity. Mostpreferably, the compounds of Formula I are useful in the treatment ofcerebral ischemia/stroke.

In still yet another aspect, this invention provides pharmaceuticalcompositions comprising at least one compound of Formula I incombination with a pharmaceutical adjuvant, carrier or diluent.

The compounds of Formula I may be prepared by various procedures such asthose illustrated herein in the examples, in the Reaction Scheme andvariations thereof which would be evident to those skilled in the art.The various prodrug compounds of Formula I may advantageously beprepared from the active drug substance of Formula II which is itselfprepared by the general procedure described in WO 98/04135 and inExample I and used as the starting material in the methods illustratedin Reaction Scheme 1. ##STR9##

The preparation of amino acid esters of 1,3,4-oxadiazol-2-(3H)-onederivatives of Formula Ia-h, is illustrated in Reaction Scheme 1. Whenit is desired to prepare the amino acid ester compounds of Formula Ia-h,the compound of Formula II is treated with a N-carbobenzyloxy amino acidof Formula IIIa-h in an anhydrous inert organic solvent such asmethylene chloride and the coupling is advantageously carried out in thepresence of 1,3-dicyclohexylcarbodiimide (DCC) to produce thecorresponding compound of Formula IVa-h. Deprotection of thecarbobenzyloxy group (Z) of Formula IVa-h with HBr in acetic acid yieldsthe HBr salt of the amino acid ester compound of Formula Ia-h.

In a preferred embodiment of the invention, the compounds have theFormula I ##STR10## wherein R¹ and R³ are hydrogen or methyl; R² is C₁₋₄alkyl optionally substituted with hydroxy, amino, methylthio, carboxyl,carboxamide, guanidino, phenyl or hydroxyphenyl; and R² and R³ may bejoined together to form a pyrrolidinyl ring; or a nontoxicpharmaceutically acceptable salt or solvate thereof. Preferably, R¹ andR³ are hydrogen and R² is C₁₋₄ alkyl or a nontoxic pharmaceuticallyacceptable salt or solvate thereof.

In another embodiment, this invention includes pharmaceuticalcompositions comprising at least one compound of Formula I incombination with a pharmaceutical adjuvant, carrier or diluent.

In still another embodiment, this invention relates to a method oftreatment or prevention of disorders responsive to opening of potassiumchannels in a mammal in need thereof, which comprises administering tosaid mammal a therapeutically effective amount of a compound of FormulaI or a nontoxic pharmaceutically acceptable salt, solvate or hydratethereof.

In yet another embodiment, this invention relates to a method fortreating ischemia, convulsions, epilepsy, asthma, irritable bowelsyndrome, migraine, traumatic brain injury, spinal cord injury, male andfemale sexual dysfunction, urinary incontinence and especially stroke ina mammal in need thereof, which comprises administering to said mammal atherapeutically effective amount of a compound of Formula I or anontoxic pharmaceutically acceptable salt, solvate or hydrate thereof.

Biological Activity

Potassium (K⁺) channels are structurally and functionally diversefamilies of K⁺ -selective channel proteins which are ubiquitous incells, indicating their central importance in regulating a number of keycell functions [Rudy, B., Neuroscience, 25, pp. 729-749 (1988)]. Whilewidely distributed as a class, K⁺ channels are differentiallydistributed as individual members of this class or as families.[Gehlert, D. R., et al., Neuroscience, 52, pp. 191-205 (1993)]. Ingeneral, activation of K⁺ channels in cells, and particularly inexcitable cells such as neurons and muscle cells, leads tohyperpolarization of the cell membrane, or in the case of depolarizedcells, to repolarization. In addition to acting as an endogenousmembrane voltage clamp, K⁺ channels can respond to important cellularevents such as changes in the intracellular concentration of ATP or theintracellular concentration of calcium (Ca²⁺). The central role of K⁺channels in regulating numerous cell functions makes them particularlyimportant targets for therapeutic development. [Cook, N. S., Potassiumchannels: Structure, classification, function and therapeutic potential.Ellis Horwood, Chinchester (1990)]. One class of K+ channels, thelarge-conductance Ca²⁺ -activated K⁺ channels (BK or BK channels), isregulated by transmembrane voltage, intracellular Ca²⁺, and a variety ofother factors such as the phosphorylation state of the channel protein.[Latorre, R., et al., Ann. Rev. Physiol., 51, pp. 385-399 (1989)]. Thelarge, single channel-conductance (generally>150 pS) and high degree ofspecificity for K⁺ of BK channels indicates that small numbers ofchannels could profoundly affect membrane conductance and cellexcitability. Additionally, the increase in open probability withincreasing intracellular Ca²⁺ indicates involvement of BK channels inthe modulation of Ca²⁺ -dependent phenomena such as secretion andmuscular contraction. [Asano, M., et al., J. Pharmacol. Exp. Ther., 267,pp.1277-1285 (1993)].

Openers of BK channels exert their cellular effects by increasing theopen probability of these channels [McKay, M. C., et al., J.Neurophysiol., 71, pp. 1873-1882 (1994); and Olesen, S. -P., Exp. Opin.Invest. Drugs, 3, pp.1181-1188 (1994)]. This increase in the opening ofindividual BK channels collectively results in the hyperpolarization ofcell membranes, particularly in depolarized cells, produced bysignificant increases in whole-cell BK-mediated conductance.

The ability of the compound of Example 1 to open BK channels andincrease whole-cell outward (K⁺) BK-mediated currents was assessed undervoltage-clamp conditions by determining their ability to increase clonedmammalian (mSlo or hSlo) BK-mediated outward current heterologouslyexpressed in Xenopus oocytes [Butler, A., et al., Science, 261, pp.221-224 (1993); and Dworetzky, S. I., et al., Mol. Brain Res., 27,pp.189-193 (1994)]. The two BK constructs employed represent nearlystructurally identical homologous proteins, and have proven to bepharmacologically identical in our tests. To isolate BK current fromnative (background, non-BK) current, the specific and potent BKchannel-blocking toxin iberiotoxin (IBTX) [Galvez, A., et al., J. Biol.Chem, 265, pp. 11083-11090 (1990)] was employed at a supramaximalconcentration (50 nM). The relative contribution of BK channels currentto total outward current was determined by subtraction of the currentremaining in the presence of IBTX (non-BK current) from the currentprofiles obtained in all other experimental conditions (control, drug,and wash). It was determined that at the tested concentration thecompound profiled did not effect non-BK native currents in the oocytes.The compound of Example 1 was shown in at least 5 oocytes at aconcentration of 1 μM to increase BK current to 126% of control ofIBTX-sensitive current. Recordings were accomplished using standardtwo-electrode voltage clamp techniques [Stuhmer, W., et al., Methods inEnzymology, 207, pp. 319-339 (1992)]; voltage-clamp protocols consistedof 500-750 ms duration step depolarizations from a holding potential of-60 mV to +140 mV in 20 mV steps. The experimental media (modifiedBarth's solution) consisted of (in mM): NaCl (88), NaHCO₃ (2.4), KCl(1.0), HEPES (10), MgSO₄ (0.82), Ca(NO₃)₂ (0.33), CaCl₂ (0.41); pH 7.5.

A rapid screen to determine the ability of prodrugs to hydrolyze andrelease the drug (compound of Example 1) is conducted as follows. A 1mg/mL stock solution of the prodrug is prepared in distilled water oracetonitrile or PEG-400. Plasma from freshly collected rat or humanblood is used in this assay. To 1 mL of plasma at 37° C. was added 10 μLof stock solution of prodrug and mixed gently. Immediately after themixing, 100 μL of plasma was removed and quenched with 300 μL ofacetontrile (Zero time sample). Samples were also obtained at 30 minutesand quenched immediately. The quenched samples were centrifuged toobtain a clear supernatant for analysis. The stock solution, T=0 andT=30 samples were analyzed by a HPLC assay that separates the drug fromthe prodrug. Based on the relative peak areas of prodrug and drug inthese samples, different prodrugs are characterized as fast, moderateand slow release agents. For example, in this model, the compound ofExample 9 was dissolved in PEG-400 at a concentration of 1 mg/mL andincubated at 10 μg/mL in fresh rat plasma at 37° C. Analysis of thesolution 5 minutes after incubation indicated conversion of the compoundof Example 9 to the compound of Example 1.

To determine the ability of the compounds of the present invention toreduce cell loss resulting from neuronal ischemia, a standard focalcerebral ischemia is induced by permanent occlusion of the left middlecerebral artery (MCA) and common carotid artery (CCA) with one hourocclusion of the right CCA in the Wistar rat. The surgeries areperformed using the sub-temporal approach of A. Tamura, et al., J.Cereb. Blood Flow Metab., 1, pp. 53-60, (1981) and its modifications [K.Osborne, et al., J. Neurol Neurosurg. Psychiatry, 50, pp. 402-410 (1987)and S. Menzies, et al., Neurosurgery, 31, pp. 100-107, (1992).]

The compound of Example 1 was evaluated in the focal stroke modelinvolving permanent occlusion of the left MCA (MCAO) and CCA (CCAO) andtemporary occlusion of the right CCA in the Wistar rat. This procedureresults in a reliably large neocortical infarct volume that is measuredby means of vital dye exclusion in serial slices through the brain 24hours after MCAO. In the present test, compounds were administered usingan i.v. or i.p. route of administration two hours after occlusion. Forexample, in this model the compound of Example 1 significantly reducedthe cortical infarct volume by about 18% when administered intravenously(10 μg/kg) as a single bolus two hours after middle cerebral arteryocclusion as compared to vehicle-treated (water) control.

The results of the above in vitro and in vivo tests demonstrate that thenovel 1,3,4-oxadiazol-2(3H)-one compounds of the present invention areuseful for the treatment of human disorders arising from dysfunction ofcellular membrane polarization and conductance and, preferably, areindicated for the treatment of ischemia, stroke, convulsions, epilepsy,asthma, irritable bowel syndrome, migraine, traumatic brain injury,spinal cord injury, sexual dysfunction, and urinary incontinence andother disorders sensitive to BK channel activating activity. Mostpreferably, the compounds of Formula I are useful in the treatment ofcerebral ischemia/stroke.

The compounds of Formula I or pharmaceutical compositions thereof areuseful in the treatment, alleviation or elimination of disorders orother disorders associated with the BK channels. Such disorders includeischemia, stroke, convulsions, epilepsy, asthma, irritable bowelsyndrome, migraine, traumatic brain injury, spinal cord injury, sexualdysfunction and urinary incontinence and other disorders sensitive topotassium channel openers.

For therapeutic use, the pharmacologically active compounds of Formula Iwill normally be administered as a pharmaceutical composition comprisingas the (or an) essential active ingredient at least one such compound inassociation with a solid or liquid pharmaceutically acceptable carrierand, optionally, with pharmaceutically acceptable adjuvants andexcipients employing standard and conventional techniques.

The pharmaceutical compositions include suitable dosage forms for oral,parenteral (including subcutaneous, intramuscular, intradermal andintravenous) bronchial or nasal administration. Thus, if a solid carrieris used, the preparation may be tableted, placed in a hard gelatincapsule in powder or pellet form, or in the form of a troche or lozenge.The solid carrier may contain conventional excipients such as bindingagents, fillers, tableting lubricants, disintegrants, wetting agents andthe like. The tablet may, if desired, be film coated by conventionaltechniques. If a liquid carrier is employed, the preparation may be inthe form of a syrup, emulsion, soft gelatin capsule, sterile vehicle forinjection, an aqueous or non-aqueous liquid suspension, or may be a dryproduct for reconstitution with water or other suitable vehicle beforeuse. Liquid preparations may contain conventional additives such assuspending agents, emulsifying agents, wetting agents, non-aqueousvehicle (including edible oils), preservatives, as well as flavoringand/or coloring agents. For parenteral administration, a vehiclenormally will comprise sterile water, at least in large part, althoughsaline solutions, glucose solutions and like may be utilized. Injectablesuspensions also may be used, in which case conventional suspendingagents may be employed. Conventional preservatives, buffering agents andthe like also may be added to the parenteral dosage forms. Particularlyuseful is the administration of a compound of Formula I directly inparenteral formulations. The pharmaceutical compositions are prepared byconventional techniques appropriate to the desired preparationcontaining appropriate amounts of the active ingredient, that is, thecompound of Formula I according to the invention. See, for example,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 17th edition, 1985.

The dosage of the compounds of Formula I to achieve a therapeutic effectwill depend not only on such factors as the age, weight and sex of thepatient and mode of administration, but also on the degree of potassiumchannel activating activity desired and the potency of the particularcompound being utilized for the particular disorder of diseaseconcerned. It is also contemplated that the treatment and dosage of theparticular compound may be administered in unit dosage form and that theunit dosage form would be adjusted accordingly by one skilled in the artto reflect the relative level of activity. The decision as to theparticular dosage to be employed (and the number of times to beadministered per day) is within the discretion of the physician, and maybe varied by titration of the dosage to the particular circumstances ofthis invention to produce the desired therapeutic effect.

A suitable dose of a compound of Formula I or pharmaceutical compositionthereof for a mammal, including man, suffering from, or likely to sufferfrom any condition as described herein is an amount of active ingredientfrom about 0.1 ng/kg to 10 mg/kg body weight. For parenteraladministration, the dose may be in the range of 0.1 ng/kg to 1.0 mg/kgbody weight for intravenous administration. The active ingredient willpreferably be administered either continuously or in equal doses fromone to four times a day. However, usually a small dosage isadministered, and the dosage is gradually increased until the optimaldosage for the host under treatment is determined.

However, it will be understood that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechoice of compound of be administered, the chosen route ofadministration, the age, weight, and response of the individual patient,and the severity of the patient's symptoms.

The following examples are given by way of illustration and are not tobe construed as limiting the invention in any way inasmuch as manyvariations of the invention are possible within the meaning of theinvention.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In the following examples, all temperatures are given in degreesCentigrade. Melting points were recorded on a Gallenkamp capillarymelting point apparatus temperatures are uncorrected. Proton magneticresonance (¹ H NMR) was recorded on a Bruker AC 300. All spectra weredetermined in the solvents indicated and chemical shifts are reported inδ units downfield from the internal standard tetramethylsilane (TMS) andinterproton coupling constants are reported in Hertz (Hz). Splittingpatterns are designated as follows: s, singlet; d, doublet; t, triplet;q, quartet; m, multiplet; br, broad peak; dd, doublet of doublet; bd,broad doublet; dt, doublet of triplet; bs, broad singlet; dq, doublet ofquartet. Infrared (IR) spectra using potassium bromide (KBr) weredetermined on a Perkin Elmer 781 spectrometer from 4000 cm⁻¹ to 400cm⁻¹, calibrated to 1601 cm⁻¹ absorption of a polystyrene film andreported in reciprocal centimeters (cm⁻¹). Low resolution mass spectra(MS) and the apparent molecular (MH⁺) or (M-H)- was determined on aFinnigen TSQ 7000. High resolution mass spectra was determined on aKratos MS50 in FAB mode using cesium iodide/glycerol as internalreference. The element analysis are reported as percent by weight.

The following examples illustrate procedures for the preparation ofstarting materials, intermediates and methods for the preparation ofproducts according to this invention. It should also be evident to thoseskilled in the art that appropriate substitution of both materials andmethods disclosed herein will produce the examples illustrated below andthose encompassed by the scope of this invention.

EXAMPLE 13-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one

Step A

5-[4-(Trifluoromethyl)phenyl]-1.3,4-oxadiazol-2(3H)-one

4-(Trifluoromethyl)benzoic acid hydrazide (commercially available fromMaybridge Chemicals) (5 g, 24.5 mmol) was taken up in THF (250ml)/triethylamine (2.7 ml, 26 mmol) under N₂ and1,1'-carbonyl-diimidazole (4.2 g, 26 mmol) added. The solution wasstirred for 18 h at 24° C., concentrated, and the residue was taken upin ethyl acetate, washed with 1 N HCl solution, sat'd NaHCO₃ solution,and brine prior to drying (MgSO₄). Concentration gave 5 g (89%) of thetitle compound from which a sample was recrystallized from diethylether/hexanes: mp 214-216° C. MS m/z: 231 (MH⁺).

IR (KBr) 3280, 1778, 1608, 1420, 1318, 1170, 1114 cm⁻¹ ;

¹ H NMR (DMSO-d₆) δ7.87 (2H, d, J=8.3 Hz), 7.96 (2H, d, J=8.3 Hz), 12.77(1H, br.s);

Anal. Calcd. for C₉ H₅ F₃ N₂ O₂.064 H₂ O: C, 46.74; H, 2.24; N, 12.11.

Found: C, 47.07; H, 2.10; N, 12.34.

Step B

3-[(5-Chloro-2-methoxyphenyl)methyl]-5-[4-(trifluoromethyl)-phenyl]-1,3,4-oxadiazol-2(3H)-one

5-[4-(Trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(H)-one (11.75 g, 51mmol) and 5-chloro-2-methoxybenzylbromide [N. Meanwell, et al., Bioorg.Med. Chem. Lett. 6, pp. 1641-1646 (1996)] (12.0 g, 51 mmol) and 11.2 g(81 mmol) of potassium carbonate were added to CH₃ CN (300ml) undernitrogen and potassium iodide (0.2 g, 1.2 mmol) was added. The solutionwas refluxed for 16 h, cooled, poured into water (1500 ml) and stirredvigorously. The precipitate was filtered to give a solid which wasrecrystallized from CH₃ CN to give 15.2 g (78%) of the title compound.

mp 144-145° C. MS(ESI)m/z: 385 (MH⁺).

IR (KBr) 3440, 1782, 1492, 1324, 1248, 1168 cm⁻¹ ;

¹ H NMR (300 MHz, DMSO) δ3.79 (3H, s), 4.91 (2H, s), 7.07 (1H, d, J=8.8Hz), 7.35-7.38 (2H, m), 7.88 (2H, d, J=8.4 Hz), 7.96 (2H, d, J=8.2 Hz);

Anal. Calcd. for C₁₇ H₁₂ ClF₃ N₂ O₃.0.1 H₂ O: C, 52.81; H, 3.19; N,7.25.

Found: C, 53.03; H, 3.20; N, 7.31.

Step C

3-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)-phenyl]-1,3,4-oxadiazol-2(3H)-one

3-[(5-Chloro-2-methoxyphenyl)methyl]-5-[4-(trifluoromethyl)-phenyl]-1,3,4-oxadiazol-2(3H)-one(15.2 g, 39.6 mmol) was admixed with pyridine hydrochloride (19.7 g,0.17 mol) and heated at 225° C. for 2 h. The hot solution was pouredinto 800 ml of 1 N HCl and the mixture was stirred for 10 min. The solidwas collected, washed with 1 N HCl and dried at 80° C. under vacuum toafford 13.1 g of an off-white solid. Recrystallization from acetonitrilegave 10.8 g of the title compound as fluffy needles, mp 217-218° C. MSm/z: 371 (MH⁺).

IR (KBr) 3354, 1762, 1500, 1324, 1068 cm⁻¹ ;

¹ H NMR (DMSO-d₆) δ4.98 (2H, s), 6.84 (1H, d, J=8.7 Hz), 7.20 (1H, dd,J=8.7 Hz, 2.6 Hz), 7.30 (1H, d, J=2.5 Hz), 7.89 (2H, d, J=8.6 Hz), 7.97(1H, d, J=8.6 Hz), 10.11 (1H, br.s);

Anal. Calcd. for C₁₆ H₁₀ ClF₃ N₂ O₃ : C, 51.84; H, 2.72; N, 7.56.

Found: C, 51.88; H, 2.58; N, 7.57.

EXAMPLE 23-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,glycine ester (la, R¹ =R² =R³ =H)

Step A

3-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H-one,N-carbobenzyloxyglycine ester (IVa, R¹ =R² =R³ =H)

Neat 1,3-dicyclohexylcarbodiimide (DCC) (95 mg, 0.46 mmol) was added tostirred mixture of3-[(5-chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one(170 mg, 0.48 mmol), N-carbobenzyloxyglycine (96 mg, 0.46 mmol) inanhydrous dichloromethane (5 mL) and maintained at room temperatureovernight. Reaction mixture was diluted with dichloromethane (30 mL),and cool to 0° C. for 1 hr before it was filtered. The filtrate wasevaporated and the residue was re-suspended in diethylether and thenfiltered. The filtrate was evaporated and the residue was recrystallizedtwice from diethylether/hexane to afford white needles (240 mg, 85%): mp116-1180° C.; MS m/e 562 (MH⁺).

¹ H NMR (CDCl₃) δ4.35 (d, J=6 Hz, 2H), 4.84 (s, 2H), 5.14 (s, 2H), 5.52(brd s, 1H), 7.10 (d, J=8.7 Hz, 1H), 7.30-7.40 (m, 6H), 7.51 (d, J=2.4Hz, 1H), 7.68 (d, J=8.4 Hz, 2H), 7.89 (d, J=8.4 Hz, 2H).

Anal. calcd. for C₂₆ H₁₉ ClF₃ N₂ O₆ : C, 55.58; H, 3.41; N, 7.48.

Found: C, 55.56; H, 3.46; N, 7.41.

Step B

3-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,glycine ester (la. R¹ =R² =R³ =H)

A mixture of3-[(5-chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,N-carbobenzyloxyglycine ester (IVa) (237 mg, 0.42 mmol) and HBr (0.65mL, 30% in acetic acid, 3.25 mmol) was stirred in acetic acid (3 mL) atroom temperature for 2 hr. The resulting reaction mixture was evaporatedand the residual solid was re-suspended in diethylether and thenfiltered, washed with diethylether and then dried in vacuo to afford awhite solid (158 mg, 88%): mp 210-211° C. (dec.); MS m/e 428 (MH⁺).

¹ H NMR (DMSO-d₆) δ4.17 (s, 2H), 4.99 (s, 2H), 7.33 (d, J=8.7 Hz,1 H),7.58 (dd, J=8.7, 2.4 Hz,1H), 7.71 (d, J=2.4 Hz,1H), 7.92 (d, J=8.4 Hz,2H), 8.00 (d, J=8.4 Hz, 2H), 8.39 (brd m, 3H).

Anal. calcd. for C₁₈ H₁₃ ClF₃ N₂ O₄.HBr: C, 42.50; H, 2.77; N, 8.26.

Found: C, 41.67; H, 2.97; N, 8.02.

EXAMPLE 33-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one, (L)-Alanine ester (lb)

The title compound was prepared using the general procedure described inExample 2. The crude product was triturated with hot ethyl acetate,cooled to room temperature and filtered. The resulting white needleswere dried in vacuo (123 mg, 68% yield): mp 214-215° C.;

¹ H NMR (DMSO-d₆) δ1.61 (d, J=7.2 Hz, 3H), 4.47 (m, 1H), 4.99 (s, 2H),7.35 (d, J=8.7 Hz,1H), 7.58 (dd, J=8.7, 2.4 Hz,1H), 7.69 (d, J=2.4 Hz,1H), 7.93 (d, J=8.4 Hz, 2H), 7.99 (d, J=8.4 Hz, 2H), 8.47 (brd m, 3H).MS m/e 442 (MH⁺).

Anal. calcd. for C₁₉ H₁₅ ClF₃ N₂ O₄.HBr: C, 43.66; H, 3.09; N, 8.04.

Found: C, 43.39; H, 2.91; N, 7.80.

EXAMPLE 43-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,α,α-Dimethyl glycine ester (lc)

The title compound was prepared by using the general procedure describedin Example 2. The crude product was recrystallized from acetone/hexanes(146 mg, 82%): mp 167-168° C.; MS m/e 456 (MH⁺).

¹ H NMR (DMSO-d₆) δ1.70 (s, 6H), 5.00 (s, 2H), 7.37 (d, J48.7 Hz, 1H),7.5 9 (dd, J=8.7, 2.4 Hz, 1H), 7.68 (d, J=2.4 Hz, 1H), 7.93 (d, J=8.7Hz, 2H), 7.99 (d, J=8.7 Hz, 2H), 8.65 (brd m, 3H).

Anal. calcd. for C₂₀ H₂₇ ClF₃ N₂ O₄.HBr: C, 44.76; H, 3.38; N, 7.83.

Found: C, 45.11; H, 3.57; N, 7.46.

EXAMPLE 53-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,(L)-Leucine ester (ld)

The title compound was prepared by using the general procedure describedin Example 2. The crude product was recrystallized from acetone/hexanes(156 mg, 78%): mp 195-1968° C.;

¹ H NMR (DMSO-d₆) δ0.96 (d, J=4.5 Hz, 6H), 1.75-1.95 (m, 3H), 4.35 (m,1H), 5.00 (dd, J=20, 12 Hz, 2H), 7.37 (d, J=8.7 Hz, 1H), 7.58 (dd,J=8.7, 2.4Hz, 1H), 7.69 (d, J=2.4 Hz, 1H), 7.93 (d, J=8.4 Hz, 2H), 7.99(d, J=8.4 Hz, 2H), 8.54 (br, 3H). MS m/e 484 (MH⁺).

Anal. calcd. for C₂₂ H₂₁ ClF₃ N₂ O₄.HBr: C, 46.79; H, 3.93; N, 7.44.

Found: C, 47.08; H, 3.93; N, 7.16.

EXAMPLE 63-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,(L)-Lysine ester (le)

The title compound was prepared by using the general procedure describedin Example 2. The crude product was triturated with hot acetone withtrace amount of ethyl acetate, cooled to room temperature, filtered andwashed with acetone and diethylether. The resulting white powder wasdried in vacuo (70%): mp 228-230° C.;

¹ H NMR (DMSO-d₆) δ1.61 (brd m, 4H), 1.96-2.08 (m, 2H), 2.81 (brd m,2H), 4.39 (brd s, 1H), 5.02 (s, 2H), 7.41 (d, J=8.7 Hz, 1H), 7.58 (dd,J=8.7, 2.4 Hz,1H), 7.69 (d, J=2.4 Hz,1H), 7.76 (brd m, 3H), 7.94 (d,J=8.7 Hz, 2H), 8.01 (d, J=8.7 Hz, 2H), 8.62 (brd m, 3H). MS m/e 499(MH⁺).

Anal. calcd. for C₂₂ H₂₂ ClF₃ N₂ O₄.2HBr: C, 39.99; H, 3.66; N, 8.48.

Found: C, 40.05; H, 3.60; N, 8.30.

EXAMPLE 73-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,(L)-Proline ester (lf)

The crude product was triturated with diethylether and trace amount ofethyl acetate and the resulting light yellow powder was dried in vacuo(180 mg, 38% for two steps). ¹ H NMR (DMSO-d₆) δ1.98-2.04 (m, 2H),2.30-2.50 (m, 2H), 3.30-3.37 (m, 2H), 4.76 (t, J=8.1 Hz, 1H), 5.02 (s,2H), 7.45 (d, J=8.7 Hz, 1H), 7.59 (dd, J=8.7, 2.4 Hz, 1H), 7.70 (d,J=2.4 Hz, 1H), 7.93 (d, J=8.4 Hz, 2H), 7.99 (d, J=8.4 Hz, 2H), 8.24 (brdm, 2H). MS m/e 468 (MH⁺).

Anal. calcd. for C₂₁ H₁₇ ClF₃ N₂ O₄.HBr: C, 45.97; H, 3.31; N, 7.66.

Found: C, 45.36; H, 3.48; N, 7.44.

EXAMPLE 83-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,N-Methyl glycine ester (lg)

The title compound was prepared using the general procedure described inExample 2. The crude product was recrystallized from ethyl acetate (38%for two steps): mp 186-188° C.;

¹ H NMR (DMSO-d₆) δ2.69 (s, 3H), 4.31 (s, 2H), 4.98 (s, 2H), 7.34 (d,J=8.7 Hz, 1H), 7.57 (dd, J=8.7, 2.7 Hz, 1H), 7.70 (d, J=2.7 Hz,1H), 7.91(d, J=8.7 Hz, 2H), 7.99 (d, J=8.7 Hz, 2H), 9.08 (brd m, 2H). MS m/e 442(MH⁺).

Anal. calcd. for C₁₉ H₁₅ ClF₃ N₂ O₄.HBr: C, 43.66; H, 3.09; N, 8.04.

Found: C, 44.27; H, 3.56; N, 7.58.

EXAMPLE 93-[(5-Chloro-2-hydroxyphenyl)methyl]-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2(3H)-one,N-Methyl (L)-Leucine ester (lh)

The title compound was prepared by using the general procedure describedin Example 2. The crude product was triturated in diethylether withtrace amount of ethyl acetate and filtered. The resulting solid wasrecrystallized from acetone/hexanes (70% for two steps): mp 145-146° C.;MS m/e 498 (MH⁺).

¹ H NMR (DMSO-d₆) δ0.97 (d, J=4.7 Hz, 6H), 1.80-2.01 (m, 3H), 2.73 (s,3H), 4.40 (brd s,1H), 5.00 (dd, J=18, 16 Hz, 2H), 7.42 (d, J=8.7 Hz,1H),7.58 (dd, J=8.7, 2.7 Hz,1H), 7.70 (d, J=2.7 Hz,1H), 7.93 (d, J=8.7 Hz,2H), 7.99 (d, J=8.7 Hz, 2H), 9.27 (brd m, 2H).

Anal. calcd. for C₂₃ H₂₃ ClF₃ N₂ O₄.HBr: C, 47.73; H, 4.18; N, 7.26.

Found: C, 47.64; H, 4.41; N, 7.19.

What is claimed:
 1. A compound of the formula ##STR11## wherein R¹ ishydrogen or methyl;R² is hydrogen; or substituted or unsubstituted C₁₋₄alkyl in which the substituent is selected from the group consisting ofhydroxy, amino, methylthio, carboxyl, carboxamide, guanidino, phenyl orhydroxyphenyl; and R³ is hydrogen or methyl; or R³ and R² are joinedtogether to form a pyrrolidinyl ring;or a nontoxic pharmaceuticallyacceptable salt or solvate thereof.
 2. The compound of claim 1 whereinR¹, R² and R³ are hydrogen or a nontoxic pharmaceutically acceptablesalt or solvate thereof.
 3. The compound of claim 1 wherein R¹ and R³are hydrogen and R² is methyl or a nontoxic pharmaceutically acceptablesalt or solvate thereof.
 4. The compound of claim 1 wherein R³ ishydrogen and R¹ and R² are methyl or a nontoxic pharmaceuticallyacceptable salt or solvate thereof.
 5. The compound of claim 1 whereinR¹ and R³ are hydrogen and R² is 2-methylpropyl or a nontoxicpharmaceutically acceptable salt or solvate thereof.
 6. The compound ofclaim 1 wherein R¹ and R³ are hydrogen and R² is aminobutyl or anontoxic pharmaceutically acceptable salt or solvate thereof.
 7. Thecompound of claim 1 wherein R¹ is hydrogen and R² and R³ are joinedtogether to form a pyrrolidinyl ring or a nontoxic pharmaceuticallyacceptable salt or solvate thereof.
 8. The compound of claim 1 whereinR¹ and R² are hydrogen and R³ is methyl or a nontoxic pharmaceuticallyacceptable salt or solvate thereof.
 9. A compound of claim 1 wherein R¹is hydrogen, R² is 2-methylpropyl and R³ is methyl or a nontoxicpharmaceutically acceptable salt or solvate thereof.
 10. Apharmaceutical composition for the treatment of disorders responsive toopeners of the large conductance calcium-activated potassium channelscomprising a therapeutically effective amount of a compound as definedin claim 1 in association with a pharmaceutically acceptable carrier ordiluent.
 11. A method for the treatment of disorders responsive toopening of the large conductance calcium-activated potassium channels ina mammal in need thereof, which comprises administering to said mammal atherapeutically effective amount of a compound as defined in claim 1.12. A method of claim 11 wherein said disorder is ischemia, stroke,convulsions, epilepsy, asthma, irritable bowel syndrome, migraine,traumatic brain injury, sexual dysfunction and urinary incontinence. 13.The method of claim 12 wherein the disorder is stroke.